Fig. 6: Epigenetic mechanisms govern HB-EGF expression during autoimmune CNS inflammation.

a, EAE development and timepoints (peak, naive) used for WGBS of astrocytes isolated by FACS. n = 4 per timepoint. b, RT–qPCR analysis of DNA-methyltransferases (DNMT) expressed in FACS-isolated astrocytes during peak EAE (n = 4) and naive (n = 3) stages. c, Quantification of DNA methylation at HIF1α and AhR binding sites in the Hbegf locus in astrocytes during peak EAE (n = 4) and naive (n = 4) stages. d, HRM with difference plot and area under the curve (AUC) of bisulfite-converted gDNA of the Hbegf promoter region from FACS-sorted astrocytes obtained from the CNS of naive or EAE mice at peak or recovery stages. n = 4 (3 replicates per sample) per timepoint. Group means were used for AUC analysis. e,f, HRM with difference plot (e) and AUC analysis (f) of methylation at HIF1α binding sites in the Hbegf promoter in mouse following stimulation with TNF-α and IL-1β ± 5-Aza. n = 4 per group. Group means were used for AUC analysis. g, 5-mC ChIP of the Hbegf promoter region in primary mouse astrocytes following stimulation with TNF-α and IL-1β ± 5-Aza. n = 5–8 per group. h, Intracellular flow cytometric analysis of HB-EGF expression by primary mouse astrocytes following stimulation with TNF-α and IL-1β ± the hypomethylating agent 5-Aza. n = 3 per group. i, Schematic of daily intranasal treatment with 5-Aza during EAE. j–m, HRM with difference plot (j), AUC analysis (k), relative levels (% of parent) of HB-EGF by flow cytometry (l) and relative mRNA expression of Hbegf by RT–qPCR in ACSA2⁺ astrocytes (m) from vehicle- or 5-Aza-treated mice during late-stage EAE. n = 4/5 per group. Group means were used for AUC analysis. n, Linear regression analysis with 95% confidence intervals of methylation in Hbegf promoter regions by AUC and relative Hbegf expression in ACSA2⁺ astrocytes obtained from vehicle- or 5-Aza-treated mice during late-stage EAE. n = 8. o, HRM analysis of methylation in the Hbegf promoter of cells obtained from whole blood of vehicle- or 5-Aza-treated mice during late-stage EAE. n = 4 per group (3 replicates per sample). Data are shown as mean ± s.d. if not indicated otherwise. Data are shown as mean ± s.e.m. in a. Two-way ANOVA with Sidak’s multiple comparisons test in b; one-way ANOVA with Dunnett’s multiple comparisons test (tested against control) in d, f, g and h; unpaired t-test in k, l and m. i.n., intranasal; Mb, megabases.

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